Project Summary Different types of immune deficiencies and cancers of the immune system which include lymphoma, different types of leukemia, multiple myeloma and others affect millions of people including children each year in the United States. Treatment options are often limited and may include risky procedures such as hematopoietic stem cell (HSC) transplantation. Ultimately, ability to differentiate hematopoietic progenitors in vitro into selected blood cell lineages including HSCs would enable more effective therapies for many different immune deficiencies and cancers. However, our knowledge of molecular mechanisms that govern hematopoiesis and immune cell differentiation is still limited. While it is difficult to study immune cell development in mammalian embryos, zebrafish has emerged as a highly advantageous system for embryonic studies. Transparent embryos are easily accessible for experimental manipulations and observations. Molecular mechanisms that regulate hematopoiesis and immune cell development are highly conserved between zebrafish and mammalian embryos. Here we have identified a novel hematopoietic site in zebrafish embryos which contains a previously unrecognized group of putative hematopoietic progenitors, pronephros associated cells (PACs). PACs are recognized by expression of transcription factors Etv2 and Scl, two known hematopoietic regulators. Our preliminary data argue that PACs can contribute to immune lineages such as macrophages and can translocate into the vasculature. However, the full lineage potential of these cells, their functional role and molecular pathways regulating their development are not known. We hypothesize that PACs represent a previously unknown type of multipotent hematopoietic progenitor cells, and contribute to immune cell lineages. The following specific aims are proposed: 1) Determine if PACs are multipotent hematopoietic progenitors and identify their lineage contributions; 2) Determine the functional role of PACs in hematopoiesis and immune system development and characterize their transcriptional profile. Lineage tracing and fate-mapping of PACs will be performed using time-lapse imaging and cell labeling approaches in zebrafish embryos to determine contribution of PACs to different hematopoietic lineages. Cell ablation will be used to deplete the PAC population, followed by the analysis of hematopoietic defects. FACS sorting approach followed by RNA-Seq will be used to define the transcriptional profile of PACs. Data obtained in this proposal will answer the key questions regarding the lineage contributions and functional role of PACs. We expect that our research will ultimately identify an alternative pathway that regulates hematopoiesis including myeloid and lymphoid progenitor development, and will lead to novel therapeutic approaches that may generate functional myeloid and lymphoid progenitors in a variety of blood and immune disorders and cancers.